Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 10 April 2019; Experimental completion date: 09 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 10 April 2019; Experimental completion date: 09 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29.07.16
Deviations:
no
Remarks:
No deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Test item identity (including alternative names): Mg-SiO, Reaction mass of MgSiO3, Si and SiO2
Product name as CoA: DMSO-E80
Physical form / color: Black powder
Batch number: E80-181130-11
Purity: 94 min wt%
Purity factor not applicable
Delivery date: 24 December 2018
Expiry date (retest date): 30 November 2019
Storage conditions: At room temperature (20 ± 5 ºC) in the dark
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information:
Species: Hannover Wistar rat
Supplier: Envigo RMS
Breeder: Envigo RMS B.V.
Total number of animals ordered: 44 males and 48 females (92 in total).
Total number of animals in the study: Groups 1 to 4 (pretest): 10 males and 10 females per group. Eight spare females were evaluated for estrous cycle to select the 10 females that formed the groups on treatment period. Groups 1 to 4 (treatment period): 10 males and 10 females, each.
Age (at treatment start): Males: 13 weeks, Females: 14 weeks
Body-weight range (at start of treatment): Males: 334-387 g, Females: 216-241 g
Allocation: On arrival, the animals were allocated at random to four treatment groups.
Method: Non-selective allocation to cages. On day 1 of pretest all animals were weighed and body weights were reviewed. No changes were made. Before treatment start and after the estrous cycle examination done at pretest, 4 females that did not show 4-5 day cycles were exchanged with spare females from the group 5 with most similar body weight and regular cycles.

Animal Care and Husbandry:
Animal Care:
Acclimatization: Five days after arrival and pretest for females. After the acclimatization period, the females were subjected to a 17-day pre-test period and males to a 9-day pre-test period.
Veterinary examination: During acclimatization at arrival (10 and 17 April 2019) and prior to treatment start (30 April 2019), the animals were examined by a veterinary surgeon. Animals without any visible signs of illness were used for treatment, except female 66 which had alopecia at pretest but was selected as its estrous cycle was regular.
Environmental enrichment program: Cellulose paper and plastic houses were supplied to reduce stress, enhance wellbeing and improve behavior.

Husbandry:
Conditions: Optimum hygienic conditions behind a barrier system: air-conditioned with 15 air changes per hour, and continuously monitored environment with ranges for temperature of 19-23 ºC and for relative humidity between 40 and 60% (occasionally the temperature reached 18 ºC and humidity, 73%) 12 hours fluorescent light/12 hours dark.
Accommodation: Cages with standard, granulated, S8-15 sawdust bedding
Premating period (4-5 animals/cage) Makrolon type IV cages
Mating period (one male and one female/cage) Makrolon type III cages
Postmating, gestation and lactation periods (individual) Makrolon type III cages

Diet:
Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum for treatment and pregnancy.
Pelleted standard Teklad 2018C rat/mouse maintenance diet ad libitum, for lactating females and pups (until sacrifice).

Water: Tap water in bottles ad libitum






Route of administration:
oral: gavage
Details on route of administration:
Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females.
The amount of administered test item was calculated according to the most recent body weight recorded.
Administration volume: 5 mL/kg
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulation:
Group 1: Vehicle control
Group 2: Test substance; 20 mg/mL
Group 3: Test substance; 66 mg/ml
Group 4: Test substance; 200 mg/ml

Dose volume: 5 mL/kg
Concentration range to be validated: 3.75 to 250 mg/mL
Frequency of dose formulation preparation: Daily and administered within between 40 minutes and 2 hours (occasionally arriving a three hours).
Storage of dose formulations: At room temperature (20 ± 5 ºC)
Stability of dose formulations: No stability information could be obtained.

Method of Preparation:
Formulation for control group: The necessary volume of vehicle was added in a suitable container.

Formulation for groups treated with test item:
A small amount of the vehicle was gradually added on the test element previously weighed in a single-use container and stirred until a homogeneous suspension was obtained. Then, the mixture was transferred to a volumetric container previously moistened with vehicle. These steps were repeated until the container containing the initial mixture was empty. The volumetric container was brought to the required volume and transferred to the final container.
The mixture was stirred on a magnetic stirrer for 10 minutes until homogeneous.
Formulations were prepared in ascending order of concentration and were maintained under agitation after preparation and during administration.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Occasions: Two, day 1 of treatment and day 29 of treatment.

Analysis of the formulations to be administered:
The formulations prepared at three different concentrations were analyzed gravimetrically to verify they were correctly prepared.
Control (vehicle) formulations were analyzed and the absence of test item was confirmed.
The test item was used as analytical standard.
Results were within ±20% of nominal value.

Sampling: 20-mL aliquots (in duplicate) were taken from the middle part of each formulation. Each aliquot was taken from the formulation freshly prepared and poured into an amber glass vial. The second aliquot was considered as a contingency sample.

Labeling: Each aliquot was labeled with the study code, test item, concentration, date of preparation, storage conditions and aliquot number.

Sample storage: Aliquot number 2 was stored frozen (-80 ± 10 ºC) and in the absence of light.

Sample disposal: Both aliquots were disposed of.

The results showed that the formulations analyzed did not deviate by more than 7% from their nominal concentration.
The analysis of control group formulations showed that no contamination was present.
Preparation of all formulations was considered correct, taking into account the acceptance ranges.






Duration of treatment / exposure:
F0 males: Two weeks prior to mating start until the day before sacrifice (five weeks of dosing).
F0 females: Two weeks prior to mating start until day 13-15 of lactation (until the day before sacrifice).
Frequency of treatment:
F0: Once daily
F1: Potential indirect exposure in utero and through milk during lactation
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control (Group 1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were chosen based on the preliminary results in a previous non-GLP 14-day Oral (Gavage) Dose-Range Toxicity Study.
As no toxicity was obtained at the administered doses in dose-range toxity study.
- The high dose was selected considering it as a limit dose to be tested.
- The intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.

Treatment Regime:
F0 Males: 2 weeks before pairing up to necropsy after a minimum of 5 weeks
F0 Females: 2 weeks before pairing, then throughout pairing and gestation until days 13 15 of lactation (until the day before sacrifice)
F1 Offspring: No direct treatment

Positive control:
No positive control
Observations and examinations performed and frequency:
VIABILITY / MORTALITY / CAGE-SIDE OBERSERVATIONS:
Animals and their cages were inspected twice daily for evidence of reaction to treatment or ill-health.

DETAILED CLINICAL SIGNS:
Animals were assessed for physical condition and behavior during handling: weekly at pretest and during treatment period including the day of necropsy.

FOOD CONSUMPTION:
Food consumption recording:
Pre-test: F0 males Days 1-8; F0 females Days 11-18
Treatment (14 days prior to mating): F0 males Days 1-8, 8-15, 15-22; F0 females Days 1-8, 8-15
Mating: -
Post-mating: F0 males Weekly.
Gestation: F0 females Days 0-6, 6-14, 14-20
Lactation: F0 females Days 1-4, 4-7, 7-13

Food consumption for female 75 (100 mg/kg/day) was recorded in periods 0-7 days and 7 14 days of gestation.

BODY WEIGHT:
Body weight recording:
Pre-test: F0 males Days 1, 6; F0 Days 1, 4, 11, 16
Treatment (14 days prior to mating): F0 males Days 1, 8, 15, F0 females Days 1, 8, 15
Mating: F0 males Weekly; F0 females Weekly
Post-mating: F0 males Days 22, 29, 34 (on sacrifice day)
Gestation: F0 females Days 0, 7, 14, 20
Lactation: F0 females Days 1, 4, 7, 13, 14 (on sacrifice day)

SENSORY REACTIVITY AND GRIP STRENGTH:
Day 30 of treatment (after dosing): 5 males and 5 females fro each dose group.
The following measurements, reflexes and responses were recorded:
- Blind reflex
- Pinna reflex
- Pupil closure reflex/Iridic light
- Proprioception (right leg) / push-off (hindlegs)
- Tail pinch response / pain response
- Startle reflex / Auditory reflex
- Air righthing

Grip strength was quantitatively measured with equipment for measuring the force of grip from BIOSEB, GT-3 model. The grip strength of the forelimbs was measured 3 times, as well as that of the hind limbs, to analyze the average value of the three occasions for hind- and forelimbs separately.

Motor Activity:
Motor activity was quantitatively measured using by automated infra-red sensor equipment AMS from Medical Instruments GmbH (FMI) and DeMeTec-Ams. For testing, designated animals were placed singly into observation cages without its pups. The test session for each animal was 1 hour with data being automatically collected at 10-minute intervals over a 60-minute period.

IN-LIFE SAMPLING AND ANALYSIS:
Samples:
Day 35 of treatment: Males - 5 animals from each group.
Days 14 or 16 of lactation: Females - 5 animals from each group.

Conditions: Males and females were deprived of food from 3-5 hours before blood extraction. Pups were removed from the litter the day before this procedure. Samples collected under light general anesthesia
Anesthetic: Isoflurane
Sample site: Retro-orbital sinus

HEMATOLOGY, PERIPHERAL BLOOD:
Sample volume: 0.5 mL/each
All samples were examined for the following characteristics:
Hematology (using EDTA as anticoagulant):
-Hematocrit (Hct)
-Hemoglobin concentration (Hb)
-Erythrocyte count (RBC)
-Absolute reticulocyte count (Retic)
-Mean cell hemoglobin (MCH)
-Mean cell hemoglobin concentration (MCHC)
-Mean cell volume (MCV)
-Red cell distribution width (RDW)
-Total leucocyte count (WBC)
-Differential leucocyte count (N: neutrophils, L: lymphocytes, E: eosinophils, B: basophils, M: monocytes, LUC: large unstained cells)
-Platelet count (Plt)

Coagulation (using citrate as anticoagulant):
- Prothrombin time (SPT)
- Activated partial thromboplastin time (SAPT)

BLOOD CHEMISTRY:
Sample volume: 0.8 mL
All samples were examined for the folllowing characteristics:
Biochemistry (using lithium heparin as anticoagulant):
-Alkaline phosphatase (ALP)
-Alanine amino-transferase (ALT)
-Aspartate amino-transferase (AST)
-Creatine kinase (CK)
-Bilirubin – total (Bili)
-Bile acids (Bi Ac)
-Urea
-Creatinine (Creat)
-Glucose (Gluc)
-Cholesterol – total (Chol)
-HDL
-LDL
-Triglycerides (Trig)
-Sodium (Na)
-Potassium (K)
-Chloride (Cl)
-Calcium (Ca)
-Phosphorus (Phos)
-Total protein (Total Prot)
-Albumin (Alb)
-Protein electrophoretogram (Alb: albumin; a1: alfa1; a2: alfa2; beta; Gamma)
-Albumin/globulin ratio (A/G Ratio)

THYROID HORMONE ANALYSIS:
Day 4 of age: 2 females per litter:
- one for T4 (serum)
- one for TSH (plasma)

Day 12 of age: Offspring: 2 males and 2 females per litter:
- two for T4 (serum); one male and one female
- two for TSH (plasma); one male and one female

At termination: All surviving F0 males and females:
- for T4 and T3 (serum)
- for TSH (plasma)

Total number of samples:
Offsrpring:
Day 4 of age : 35 for T4 and 34 for TSH
Day 13 of age: 73 for T4 and 73 for TSH

Adults:
Terminal samples : Terminal samples: 80 per parameter, 160 in total

Analysis : Samples from offspring on Day 13 of age and adult males were assessed for levels of Thyroxine (T4).
Method: Thyroxine (T4)
Bioanalysis











































Sacrifice and pathology:
NECROPSY:
F0 Males: After 5 weeks of treatment (days 35 and 36 of treatment)
F0 Females which were not pregnant: Days 24-25 after mating
F0 Females failing to produce viable litter: Day 26 after mating
F0 Female whose litters died before Day 13: On day 2 of lactation (when the offspring missed).
F0 Females killed at termination: Day 14-16 of lactation
F1 Offspring :
Selected offspring for thyroid hormone analysis – Day 4 of age
Selected offspring for thyroid hormone analysis - Day 13 of age
Remaining offspring – Day before female sacrificed

Method of Sacrifice:
F0 Animals: By intraperitoneal injection of sodium pentobarbital. Each animal was subsequently exsanguinated.
F1 Offspring: Selected for thyroid hormone sampling: Cardiac puncture. Death was assured with sodium pentobarbital injection.
All other offspring: injection of sodium pentobarbital.

Gross Pathology:
Premature decdents:
F1 Offspring: External macroscopic examination with an assessment of stomach for milk content, no thyroids to be retained.

Scheduled termination:
F0 Males:
Blood sampling required
Necropsy, macroscopic examinations and organs /tissues retained as follows:
- Males selected for FOB and clinical pathology: see Table B "Pathology procedures for the five males and the five lactating F0 females with a surviving litter per group at scheduled termination"
- Remaining males: See Table C "Pathology procedures for remaining F0 males and females per group"

F0 Females:
Blood sampling required
Necropsy, macroscopic examinations and organs /tissues retained as follows:
- The number of uterine implantation sites counted and recorded
- Terminal vaginal smear
- Females selected for FOB and clincial pathology: see Table B "Pathology procedures for the five males and the five lactating F0 females with a surviving litter per group at scheduled termination"
- Remaining females: See Table C "Pathology procedures for remaining F0 males and females per group"

F0 Females which were not pregnant and failied to litter:
Blood sampling
Necropsy, macroscopic examinations and organs/tissues retained as specified in Table C "Pathology procedures for remaining F0 males and females per group"
Number of implantation sites confirmed after the uterus was placed in an aqueous solution of ammonium sulfide.
Evaluation of mammary tissue and retained.
Terminal vaginal smear

F0 Females whose litter died before Day 13 lactation:
Blood sampling required
Necropsy, macroscopic examinations and organs/tissues retained as specified in Table C ""Pathology procedures for remaining F0 males and females per group"
Evaluation of mammary tissue and retained.
Terminal vaginal smear

F1 offspring on Day 4 of age:
Assessment of stomach for milk content. Offspring with no clinical observations discarded without examination.

F1 offspring on Day 13-15 of age:
Thyroid gland was retained from one male and one female in each litter which blood was collected for thyroid hormone analysis.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Abnormalities retained in appropriate fixative.

Organ Weights: Yes - Groups 1 - 4: Five F0 males and five lactating F0 femals with a surviving litter
Adult animals: See Table B and Table C
Data collection: For bilateral organs, left and right organs were weighed together.

In group 2 (100 mg/kg/day) organs were weighed in six females instead of five.

Fixation:
Fixatives:
Standard – 10% Neutral Buffered Formalin.
Testes and epididymides: modified Davidson’s fixative.
Eyes: Davidson’s fixative.

HISTOLOGY:
Processing (Table B): The F0 five males and five lactating females with a surviving litter selected in Groups 1 and 4 at scheduled termination.
Processing - Abnormalities: All adult animals
F0 Females which were not pregnant, failed to litter and whose litter died before Day 13 of lactation: Mammary tissue
Routine staining: 4-5 µm sections stained with hematoxylin and eosin, except testes which are also stained with periodic acid-Schiff

Pathology:
Light microscopy:
Groups 1 and 4: Five F0 males and five lactating F0 females with a surviving litter.
Data Recording
Ovaries: qualitative evaluation of one section from each ovary.
Testes: detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.





















Other examinations:
REPRODUCTIVE EVALUATIONS:
Estrous Cycles:
Dry smears (using inoculation loops):
• For 15 days before treatment (all females including spares)
• Daily from the beginning of treatment period until evidence of mating.
• On days 11-14 of lactation, including day of necropsy for females with live pups.
• On the day of necropsy for females which were not pregnant (female no. 81 at 100 mg/kg/day and female no. 86 at 330 mg/kg/day), whose litter died before Day 13 of lactation (female no. 105 at 0 mg/kg/day), and/or failed to litter (female no. 76 at 100 mg/kg/day).

Mating:
A cotton swab impregnated in distilled water was used in order to check the evidence of mating.
Paired for mating: On day 15 of treatment.
Male/female ratio: 1:1
Duration of pairing: Up to 4 days
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.

Parturition Observations and Gestation Length:
Parturition observations:
From Day 20 of pregnancy, females were checked 3 times daily for evidence of parturition.
Number of live and dead offspring was recorded during litter.
Day 1 of lactation:
When parturition ended before 07:00 am, this day was considered as Day 1 of lactation and if parturition ended after 07:00 am this day was considered as Day 0 of lactation.
Gestation length:
Time elapsing between mating and parturition.
Nursing:
The females that gave birth were checked to observe whether they nurse their offspring.

Records Made During Littering Phase:
Clinical observations: Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size: Daily from Day 1-13 of age
Sex ratio: Days 1, 4, 7 and 13 of age
Individual offspring body weights: Days 1, 4, 7 and 13 of age
Ano-genital distance: Day 1
Nipple/areolae count: Day 13 of age - male offspring; except for offspring of female no. 92, for which it was not determined.










Statistics:
Data types:
The following data types will be analyzed at each time point separately at the test facility, unless indicated otherwise:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods (males on pretest to be performed by PI)
Blood chemistry and hematology
Hormone analyses
Organ weights, both absolute and adjusted for terminal body weight
Grip strength and motor activity (to be performed by PI)
Estrous cycles (to be performed by PI)
Pre-coital interval (to be performed by the PI)
Mating performance and fertility (to be performed by PI)
Gestation length (to be performed by the PI)
Litter data (litter size, offspring survival, offspring body weight)
Ano-genital distance, average for each litter adjusted for litter average pup body weight (to be performed by the PI)
Number of nipples/areolae in male pups counted on post natal Day 13 (to be performed by the PI)

Methods:
The following comparisons were performed:
Group 1 vs. 2, 3 and 4
For categorical data, the proportion of animals was analyzed for each treated group (as appropriate) versus the control group.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. Treated groups were compared to control using Williams' test.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test.
For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.












Clinical signs:
no effects observed
Description (incidence and severity):
Dark feces were recorded in males and females from day 2 of treatment to the end of study at 1000 mg/kg/day and from day 5 of treatment to the end of study at 330 mg/kg/day. These signs were considered related to the test item color.
No other signs considered related to treatment with the test item were observed during the study.
Mortality:
no mortality observed
Description (incidence):
No mortality has been recorded.
Female no. 76 at 100 mg/kg/day was sacrificed on gestation day 25 and females 81 at 100 mg/kg/day and 86 at 330 mg/kg/day on gestation day 24 as they did not give birth. Females nos. 81 and 86 were found to be not pregnant and female no. 76 had two fetuses in the uterus (failed to litter).
Female no. 105 at 0 mg/kg/day was sacrificed on day 2 of lactation after losing its litter.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Lower body-weight gain was observed in males on the following treatment periods: days 8-15 and 29-34 at 330 mg/kg/day and days 29-34 at 1000 mg/kg/day. Even though these differences are statistically significant, the percentage with respect to the control group is lower than 10% and therefore the differences are considered of no toxicological relevance.
No relevant differences were observed in females at any dose treated group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Treatment with test item resulted in no effects on food consumption.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Although no dose-effect relation was observed in hematology or coagulation, significantly lower mean values compared to Control were recorded in prothrombin time in males at 1000 mg/kg/day after 34 treatment days (21.6 sec vs 22.7 sec). This difference with respect to Control is minor and values lower than 21.6 sec were observed at 330 mg/kg/day; therefore this is attributed to the lower standard deviation recorded at 1000 mg/kg/day.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry after 5 weeks of treatment in males and on day 14 or 16 of lactation in females revealed lower values in alanine transferase, aspartate aminotransferase and biliary acids at all treated groups and lower creatinine kinase values at 100 and 1000 mg/kg/day - see summary of results in any other information on results section

In addition, lower creatinine levels and higher triglyceride values were recorded in males from all treated groups. The differences in glucose values recorded in females from the high dose group are attributed to the high SD observed in control animals.

There was a higher increase in sodium and chloride values in males and females at 330 and 1000 mg/kg/day with respect to control animals. Lower values in phosphor and calcium were also observed in females at 1000 mg/kg/day. However, these differences were considered not relevant, as no dose-effect relation was observed and given that these mean values were within the normal range of variability observed in animals under similar conditions.

Statistically significant differences observed in protein electrophoretogram at the test item administered groups compared to Controls were considered minimal and were therefore attributed to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity assessment conducted during week 5 in males and between days 7-8 of lactation in females revealed no relevant findings. Male no. 2 failed blink and pupillary closure reflexes of left eye.
There were no relevant effects observed in the grip strength in females. However, males treated at 1000 mg/kg/day had values higher than the control group in both limbs. The differences observed were devoid of any toxicological significance.
The motor activity assessment conducted during week 5 in males and between days 7-8 of lactation in females showed no dose-related significant differences in the test-item-administered females at any time period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A trend towards an increase in adrenal and thyroid weights (absolute and relative values) was observed in males.
Absolute and relative spleen weight in females was higher than in Controls in all test-item-administered groups (100, 330 and 1000 mg/kg/day), as well as thymus weight at 330 and 1000 mg/kg/day. Differences were not dose-related or statistically significant.
The statistical significance recorded in epididymis at 1000 mg/kg/day is considered to be incidental and unrelated to the test item.
- see summary of results in any other information on results section
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic Pathology: All the gross findings are considered to be incidental and unrelated to the test item.
Dilated renal pelvis was observed in two males and two females at 0 mg/kg/day and one male and three females at 330 mg/kg/day. Three of them (male no. 5, female nos. 105 and 84) showed also changes in lymph nodes such as red mandibular lymph node, enlarged mandibular lymph node and red mesenteric lymph node, respectively.
Enlarged mandibular lymph node, dark mandibular lymph node and red mesenteric lymph node were observed in male 2 at 0 mg/kg/day, female 79 at 100 mg/kg/day and female 100 at 1000 mg/kg/day.
In addition, at 100 mg/kg/day two females showed cecum with reddish mucosa and male 18 had ileum attached to the cecum with reddish fluid inside. At 330 mg/kg/day, male 23 showed left testis absent with small epididymis and male 30 had diaphragmatic hernia with a portion of liver in the thorax cavity.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All the histological findings at termination were considered to be incidental and unrelated to treatment.
There was no histological correlate for the increased adrenal gland weights in males given 1000 mg/kg compared with controls.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES:
Analyses of samples for thyroxine (T4) obtained from F0 study males and the F1 offspring on Day 13 of age (male and female pups) did not reveal any differences attributable to treatment and there was no evidence of growth effects on the offspring supporting the conclusion that there is no effect on thyroid function.

ESTROUS CYCLES, PRE-COITAL INTERVAL, GESTATION LENGTH, MATING PERFORMANC AND FERTILITY AND GESTATION INDICES:
Before treatment start, all females showed regular cycles. However, one female (no. 105) at 0 mg/kg/day, one female (no. 76) at 100 mg/kg/day and one female (no. 82) at 330 mg/kg/day showed irregular cycles during treatment. Given the frequency observed, it is considered not to have any toxicological relevance.
At the end of lactation phase, all reproductive females showed diestrus with the exception of two females (nos. 63 and 68) at 0 mg/kg/day and one female at 1000 mg/kg/day (no. 92) which showed proestrus on day 14 of lactation and one female at 100 mg/kg/day (no. 73), which started cycling on day 12 of lactation.
There was no effect of treatment on the pre-coital interval; all animals mated within four days of mating at the first estrus.
The duration of the gestation of females treated with the test item was similar to that of the control group. Female no.76, which did not litter, was sacrificed on day 25 of pregnancy and showed two fetuses in the uterus.
Mating performance was 100% in all test-item groups and conception and fertility were 90% at 330 and 1000 mg/kg/day and 100% at 0 and 100 mg/kg/day. This slight difference is not considered relevant as it is within the historical control data.

F1 SURVIVAL INDICES, LITTER SIZE AND SEX RATIO:
No relevant differences were observed in post implantation losses, live birth index, and viability index on days 4, 7 and 13 of lactation. However the mean of total of pups littered (dead and alive) and the mean of live pups on days 1 and 4 at 330 and 1000 mg/kg/day were slightly lower than in the control group. The differences were only significant at 1000 mg/kg/day.
Litter no 105 from the control group was sacrificed on day 2 of lactation after losing its litter.
No test-item-related effects were observed on sex ratio.

OFFSPRING CLINICAL SIGNS:
There were no clinical signs observed among the F1 litters that could be clearly attributable to parental treatment with the test item.
Clinical signs recorded in some of the offspring were related with the lack of maternal care (such as little or no milk present in the stomach or pallor), not due to a direct test-item effect in pups.
In the control group, litter no 66 had three pups with changes on tail due to maternal biting: pup 1 with partial absence of the appendage, pup 2 with black spot at distal region and pup 9 with hind paw wound. Pup 7 from female 70 also had dark nose.
Little or no milk present in stomach was observed in pup 9 (litter 68) at the age of 4 and 5 days and in pup 1 (litter 105) on day 1. This last pup was also pale, thin and cold to the touch and finally it was devoured.
At 100 mg/kg/day, little or no milk present in stomach was observed in pup 9 (litter 73) on day 1 of age and in pup 7 (litter 72) on days 7 to 9 of age, which had been devoured.
At 330 mg/kg/day, little or no milk present in stomach was observed in pup 15 (litter 82) on day 1 of lactation and finally it was devoured.
At 1000 mg/kg/day, pup 1 (litter 96) was devoured after showing thinness on day 2 of lactation.

OFFSPRING BODY WEIGHTS:
There was no effect in the offspring body weight at 100 mg/kg/day.
There was a dose-related increase in offspring body weight of female and male pups at the doses of 330 and 1000 mg/kg/day. The differences observed are statistically significant.

ANO-GENITAL DISTANCE AND NIPPLE AREOLAE:
There was no effect in the mean ano-genital distance and weight adjusted to body weight in males. However, in female offspring, the adjusted ano-genital distance at 1000 mg/kg/day was slightly lower due to the higher body weight offspring observed.
There were no effects in male nipples.

OFFSPRING MACROPATHOLOGY:
There were no macroscopic findings attributable to maternal treatment with test item on the offspring that died prior to the scheduled termination or among the offspring killed on days 4 or 13-15 of age.
Two pups (2M and 9F) from litter 66 (Control group) had absence of distal part of tail. This observation was previously noted as clinical sign.
























Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.
Dose descriptor:
NOAEL
Remarks:
reproductive/developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect of toxicological relevance on estrous cycle, pre-coital interval, mating performance, fertility or gestation length, or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.
Key result
Critical effects observed:
no

Blood Chemistry:

Group

ALT

(U/L)

Males

AST

 (U/L)

Males

CK

(U/L)

Males

 Bilid Acid

 (µmol/L)

 Males

 ALT

 (U/L)

 Females

 AST

 (U/L)

 Females

 CK

 (U/L)

 Females

 Bilid Acid

 (µmol/L)

 Females

0 mg/kg/day

59

78

215

52.1

85

184

234

85.5

100 mg/kg/day

46

64*

153

40.2

60

103

190

41.0

330 mg/kg/day

45

58*

183

31.8

76

163

237

75.1

1000 mg/kg/day

47

66*

133*

39.5

50

86

155

53.1

Organ weights:

Group

Adrenals (%)

Relative to BW

Thyroids and Parathyroids (%)

Relative to BW

0 mg/kg/day

0.0151

 0.0050

100 mg/kg/day

0.0158

 0.0057

330 mg/kg/day

0.0166

 0.0063*

1000 mg/kg/day

0.0181**

 0.0060*

Group

Spleen (%)

Relative to BW

Thymus (%)

Relative to BW

0 mg/kg/day

0.200

0.0575

100 mg/kg/day

0.224

0.0616

330 mg/kg/day

0.223

0.1085

1000 mg/kg/day

0.231

0.0741

Conclusions:
The effects of oral (gavage) administration of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide to Wistar rats receiving 100, 330 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:
The No Adverse Observed Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:The No Adverse Observed Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect of toxicological relevance on estrous cycle, pre-coital interval, mating performance, fertility or gestation length, or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Executive summary:

Introduction:

The purpose of this study was to assess the general systemic toxic potential of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by gavage administration for at least 5-8 weeks.

Three groups of 10 male and 10 female rats receivedReaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxideat the doses of 100, 330 and 1000 mg/kg/day. Males were treated continuously for two weeks before pairing up to necropsy, after 35-36 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 13-15 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13-15 of lactation (the day before the corresponding female was killed). F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, Arachis oil.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, thyroid hormone, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight, ano-genital distance, nipple/areolae count and macropathology were also assessed for all offspring.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, thyroid hormone, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight, ano-genital distance, nipple/areolae count and macropathology were also assessed for all offspring.

Results:

The study results can be summarized as follows:

-         Preparation of all formulations was considered correct considering the acceptance ranges as formulation results demonstrated that all formulations were within the necessary acceptance range for the nominal concentration and coefficient of variation.

-         No mortality was recorded.

-         Administration of test item at 100, 330 or 1000 mg/kg/day was considered not to have caused any changes of toxicological relevance in clinical condition, body weight, food consumption, motor activity, sensory reactivity or grip strength.

-         Black feces due to the test item color were recorded during treatment in all animals treated at 330 and 1000 mg/kg/day.

-         There were no relevant effects on hematology or coagulation.

-         Although a dose-response effect was not recorded, the hepatic enzymes, CK and biliary acid values of the animals treated with the test item showed significant differences, with mean values lower than those seen in the Control group. In addition, mean sodium and chloride values higher than those observed in Control were also found in animals at 330 and 1000 mg/kg/day.

-         T4 analyses of samples in Main study males and F1 offspring on day 13 did not reveal any differences that could be attributable to treatment.

-         There were no macroscopic findings that could be considered test-item-related.

-         Adrenal and thyroid weight increase was observed in males at 330 and 1000 mg/kg/day. There was an increase in thymus and spleen weights in females from all treated groups.

-         Histopathology revealed no changes that were considered related to treatment withthe test item.

-         All females allocated to the study showed regular 4-day estrous cycles before treatment. The three females at 0, 100 and 330 mg/kg/day, which showed irregular cycles during treatment, achieved their pregnancy. At termination, 2/9 females at 0 mg/kg/day, 1/8 at 330 mg/kg/day and 1/10 at 1000 mg/kg/day had recovered estrous cycle.

-         No effect was observed in relation to the number of implantations,post implantation, survival indices,litter size or sex ratio.

-         Offspring body weight increased at 330 and 1000 mg/kg/day affecting both sexes. No differences in clinical signs, ano-genital distances, nipples count orexternal macroscopic examination at sacrificewere observed.

Conclusion:

The effects of oral (gavage) administration of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide to Wistar rats receiving 100, 330 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:

The No Adverse Observed Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:The No Adverse Observed Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect of toxicological relevance on estrous cycle, pre-coital interval, mating performance, fertility or gestation length, or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29.07.16
Deviations:
no
Remarks:
No deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicon
EC Number:
231-130-8
EC Name:
Silicon
Cas Number:
7440-21-3
Molecular formula:
Si
IUPAC Name:
Silicon
Constituent 2
Chemical structure
Reference substance name:
Magnesium silicate
EC Number:
237-413-2
EC Name:
Magnesium silicate
Cas Number:
13776-74-4
Molecular formula:
H2O3Si.Mg
IUPAC Name:
magnesium(2+) ion oxosilanebis(olate), crystalline
Constituent 3
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
Silicon dioxide, amorphous
impurity 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Water
impurity 2
Chemical structure
Reference substance name:
Carbon
EC Number:
231-153-3
EC Name:
Carbon
Cas Number:
7440-44-0
Molecular formula:
C
IUPAC Name:
Carbon
impurity 3
Reference substance name:
Metallic impurities (Na and S)
IUPAC Name:
Metallic impurities (Na and S)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Test item identity (including alternative names): Mg-SiO, Reaction mass of MgSiO3, Si and SiO2
Product name as CoA: DMSO-E80
Physical form / color: Black powder
Batch number: E80-181130-11
Purity: 94 min wt%
Purity factor not applicable
Delivery date: 24 December 2018
Expiry date (retest date): 30 November 2019
Storage conditions: At room temperature (20 ± 5 ºC) in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information:
Species: Hannover Wistar rat
Supplier: Envigo RMS
Breeder: Envigo RMS B.V.
Total number of animals ordered: 44 males and 48 females (92 in total).
Total number of animals in the study: Groups 1 to 4 (pretest): 10 males and 10 females per group. Eight spare females were evaluated for estrous cycle to select the 10 females that formed the groups on treatment period. Groups 1 to 4 (treatment period): 10 males and 10 females, each.
Age (at treatment start): Males: 13 weeks, Females: 14 weeks
Body-weight range (at start of treatment): Males: 334-387 g, Females: 216-241 g
Allocation: On arrival, the animals were allocated at random to four treatment groups.
Method: Non-selective allocation to cages. On day 1 of pretest all animals were weighed and body weights were reviewed. No changes were made. Before treatment start and after the estrous cycle examination done at pretest, 4 females that did not show 4-5 day cycles were exchanged with spare females from the group 5 with most similar body weight and regular cycles.

Animal Care and Husbandry:
Animal Care:
Acclimatization: Five days after arrival and pretest for females. After the acclimatization period, the females were subjected to a 17-day pre-test period and males to a 9-day pre-test period.
Veterinary examination: During acclimatization at arrival (10 and 17 April 2019) and prior to treatment start (30 April 2019), the animals were examined by a veterinary surgeon. Animals without any visible signs of illness were used for treatment, except female 66 which had alopecia at pretest but was selected as its estrous cycle was regular.
Environmental enrichment program: Cellulose paper and plastic houses were supplied to reduce stress, enhance wellbeing and improve behavior.

Husbandry:
Conditions: Optimum hygienic conditions behind a barrier system: air-conditioned with 15 air changes per hour, and continuously monitored environment with ranges for temperature of 19-23 ºC and for relative humidity between 40 and 60% (occasionally the temperature reached 18 ºC and humidity, 73%) 12 hours fluorescent light/12 hours dark.
Accommodation: Cages with standard, granulated, S8-15 sawdust bedding
Premating period (4-5 animals/cage) Makrolon type IV cages
Mating period (one male and one female/cage) Makrolon type III cages
Postmating, gestation and lactation periods (individual) Makrolon type III cages

Diet:
Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum for treatment and pregnancy.
Pelleted standard Teklad 2018C rat/mouse maintenance diet ad libitum, for lactating females and pups (until sacrifice).

Water: Tap water in bottles ad libitum






Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Oral Gavage: Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females.
The amount of administered test item was calculated according to the most recent body weight recorded.
Administration volume: 5 mL/kg

PREPARATION OF DOSING SOLUTIONS:
Formulation:
Group 1: Vehicle control
Group 2: Test substance; 20 mg/mL
Group 3: Test substance; 66 mg/ml
Group 4: Test substance; 200 mg/ml

Dose volume: 5 mL/kg
Concentration range to be validated: 3.75 to 250 mg/mL
Frequency of dose formulation preparation: Daily and administered within between 40 minutes and 2 hours (occasionally arriving a three hours).
Storage of dose formulations: At room temperature (20 ± 5 ºC)
Stability of dose formulations: No stability information could be obtained.

Method of Preparation:
Formulation for control group: The necessary volume of vehicle was added in a suitable container.

Formulation for groups treated with test item:
A small amount of the vehicle was gradually added on the test element previously weighed in a single-use container and stirred until a homogeneous suspension was obtained. Then, the mixture was transferred to a volumetric container previously moistened with vehicle. These steps were repeated until the container containing the initial mixture was empty. The volumetric container was brought to the required volume and transferred to the final container.
The mixture was stirred on a magnetic stirrer for 10 minutes until homogeneous.
Formulations were prepared in ascending order of concentration and were maintained under agitation after preparation and during administration.
Details on mating procedure:
A cotton swab impregnated in distilled water was used in order to check the evidence of mating.
Paired for mating: On day 15 of treatment.
Male/female ratio: 1:1
Duration of pairing: Up to 4 days
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Occasions: Two, day 1 of treatment and day 29 of treatment.

Analysis of the formulations to be administered:
The formulations prepared at three different concentrations were analyzed gravimetrically to verify they were correctly prepared.
Control (vehicle) formulations were analyzed and the absence of test item was confirmed.
The test item was used as analytical standard.
Results were within ±20% of nominal value.

Sampling: 20-mL aliquots (in duplicate) were taken from the middle part of each formulation. Each aliquot was taken from the formulation freshly prepared and poured into an amber glass vial. The second aliquot was considered as a contingency sample.

Labeling: Each aliquot was labeled with the study code, test item, concentration, date of preparation, storage conditions and aliquot number.

Sample storage: Aliquot number 2 was stored frozen (-80 ± 10 ºC) and in the absence of light.

Sample disposal: Both aliquots were disposed of.

The results showed that the formulations analyzed did not deviate by more than 7% from their nominal concentration.
The analysis of control group formulations showed that no contamination was present.
Preparation of all formulations was considered correct, taking into account the acceptance ranges.






Duration of treatment / exposure:
F0 males: Two weeks prior to mating start until the day before sacrifice (five weeks of dosing).
F0 females: Two weeks prior to mating start until day 13-15 of lactation (until the day before sacrifice).
Frequency of treatment:
F0: Once daily
F1: Potential indirect exposure in utero and through milk during lactation
Details on study schedule:
Treatment Regime:
F0 Males: 2 weeks before pairing up to necropsy after a minimum of 5 weeks
F0 Females: 2 weeks before pairing, then throughout pairing and gestation until days 13 15 of lactation (until the day before sacrifice)
F1 Offspring: No direct treatment

See study design flow chart in illustration section.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control (Group 1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were chosen based on the preliminary results in a previous non-GLP 14-day Oral (Gavage) Dose-Range Toxicity Study.
As no toxicity was obtained at the administered doses in dose-range toxity study.
- The high dose was selected considering it as a limit dose to be tested.
- The intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.
Positive control:
No positive control

Examinations

Parental animals: Observations and examinations:
VIABILITY / MORTALITY / CAGE-SIDE OBERSERVATIONS:
Animals and their cages were inspected twice daily for evidence of reaction to treatment or ill-health.

DETAILED CLINICAL SIGNS:
Animals were assessed for physical condition and behavior during handling: weekly at pretest and during treatment period including the day of necropsy.

FOOD CONSUMPTION:
Food consumption recording:
Pre-test: F0 males Days 1-8; F0 females Days 11-18
Treatment (14 days prior to mating): F0 males Days 1-8, 8-15, 15-22; F0 females Days 1-8, 8-15
Mating: -
Post-mating: F0 males Weekly.
Gestation: F0 females Days 0-6, 6-14, 14-20
Lactation: F0 females Days 1-4, 4-7, 7-13

Food consumption for female 75 (100 mg/kg/day) was recorded in periods 0-7 days and 7 14 days of gestation.

BODY WEIGHT:
Body weight recording:
Pre-test: F0 males Days 1, 6; F0 Days 1, 4, 11, 16
Treatment (14 days prior to mating): F0 males Days 1, 8, 15, F0 females Days 1, 8, 15
Mating: F0 males Weekly; F0 females Weekly
Post-mating: F0 males Days 22, 29, 34 (on sacrifice day)
Gestation: F0 females Days 0, 7, 14, 20
Lactation: F0 females Days 1, 4, 7, 13, 14 (on sacrifice day)

SENSORY REACTIVITY AND GRIP STRENGTH:
Day 30 of treatment (after dosing): 5 males and 5 females fro each dose group.
The following measurements, reflexes and responses were recorded:
- Blind reflex
- Pinna reflex
- Pupil closure reflex/Iridic light
- Proprioception (right leg) / push-off (hindlegs)
- Tail pinch response / pain response
- Startle reflex / Auditory reflex
- Air righthing

Grip strength was quantitatively measured with equipment for measuring the force of grip from BIOSEB, GT-3 model. The grip strength of the forelimbs was measured 3 times, as well as that of the hind limbs, to analyze the average value of the three occasions for hind- and forelimbs separately.

Motor Activity:
Motor activity was quantitatively measured using by automated infra-red sensor equipment AMS from Medical Instruments GmbH (FMI) and DeMeTec-Ams. For testing, designated animals were placed singly into observation cages without its pups. The test session for each animal was 1 hour with data being automatically collected at 10-minute intervals over a 60-minute period.

IN-LIFE SAMPLING AND ANALYSIS:
Samples:
Day 35 of treatment: Males - 5 animals from each group.
Days 14 or 16 of lactation: Females - 5 animals from each group.

Conditions: Males and females were deprived of food from 3-5 hours before blood extraction. Pups were removed from the litter the day before this procedure. Samples collected under light general anesthesia
Anesthetic: Isoflurane
Sample site: Retro-orbital sinus

HEMATOLOGY, PERIPHERAL BLOOD:
Sample volume: 0.5 mL/each
All samples were examined for the following characteristics:
Hematology (using EDTA as anticoagulant):
-Hematocrit (Hct)
-Hemoglobin concentration (Hb)
-Erythrocyte count (RBC)
-Absolute reticulocyte count (Retic)
-Mean cell hemoglobin (MCH)
-Mean cell hemoglobin concentration (MCHC)
-Mean cell volume (MCV)
-Red cell distribution width (RDW)
-Total leucocyte count (WBC)
-Differential leucocyte count (N: neutrophils, L: lymphocytes, E: eosinophils, B: basophils, M: monocytes, LUC: large unstained cells)
-Platelet count (Plt)

Coagulation (using citrate as anticoagulant):
- Prothrombin time (SPT)
- Activated partial thromboplastin time (SAPT)

BLOOD CHEMISTRY:
Sample volume: 0.8 mL
All samples were examined for the folllowing characteristics:
Biochemistry (using lithium heparin as anticoagulant):
-Alkaline phosphatase (ALP)
-Alanine amino-transferase (ALT)
-Aspartate amino-transferase (AST)
-Creatine kinase (CK)
-Bilirubin – total (Bili)
-Bile acids (Bi Ac)
-Urea
-Creatinine (Creat)
-Glucose (Gluc)
-Cholesterol – total (Chol)
-HDL
-LDL
-Triglycerides (Trig)
-Sodium (Na)
-Potassium (K)
-Chloride (Cl)
-Calcium (Ca)
-Phosphorus (Phos)
-Total protein (Total Prot)
-Albumin (Alb)
-Protein electrophoretogram (Alb: albumin; a1: alfa1; a2: alfa2; beta; Gamma)
-Albumin/globulin ratio (A/G Ratio)

THYROID HORMONE ANALYSIS:
Day 4 of age: 2 females per litter:
- one for T4 (serum)
- one for TSH (plasma)

Day 12 of age: Offspring: 2 males and 2 females per litter:
- two for T4 (serum); one male and one female
- two for TSH (plasma); one male and one female

At termination: All surviving F0 males and females:
- for T4 and T3 (serum)
- for TSH (plasma)

Total number of samples:
Offsrpring:
Day 4 of age : 35 for T4 and 34 for TSH
Day 13 of age: 73 for T4 and 73 for TSH

Adults:
Terminal samples : Terminal samples: 80 per parameter, 160 in total

Analysis : Samples from offspring on Day 13 of age and adult males were assessed for levels of Thyroxine (T4).
Method: Thyroxine (T4)
Bioanalysis

PARTURITION OBSERVATIONS AND GESTATION LENGTH
Parturition observations:
From Day 20 of pregnancy, females were checked 3 times daily for evidence of parturition.
Number of live and dead offspring was recorded during litter.
Day 1 of lactation:
When parturition ended before 07:00 am, this day was considered as Day 1 of lactation and if parturition ended after 07:00 am this day was considered as Day 0 of lactation.
Gestation length:
Time elapsing between mating and parturition.
Nursing:
The females that gave birth were checked to observe whether they nurse their offspring.























Oestrous cyclicity (parental animals):
Estrous Cycles:
Dry smears (using inoculation loops):
• For 15 days before treatment (all females including spares)
• Daily from the beginning of treatment period until evidence of mating.
• On days 11-14 of lactation, including day of necropsy for females with live pups.
• On the day of necropsy for females which were not pregnant (female no. 81 at 100 mg/kg/day and female no. 86 at 330 mg/kg/day), whose litter died before Day 13 of lactation (female no. 105 at 0 mg/kg/day), and/or failed to litter (female no. 76 at 100 mg/kg/day).
Sperm parameters (parental animals):
Light Microscopy:
Testes: detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Also see gross pathology details (postmortem examinations - parental aninmals section).
Litter observations:
Records Made During Littering Phase:
Clinical observations: Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size: Daily from Day 1-13 of age
Sex ratio: Days 1, 4, 7 and 13 of age
Individual offspring body weights: Days 1, 4, 7 and 13 of age
Ano-genital distance: Day 1
Nipple/areolae count: Day 13 of age - male offspring; except for offspring of female no. 92, for which it was not determined.
Postmortem examinations (parental animals):
NECROPSY:
F0 Males: After 5 weeks of treatment (days 35 and 36 of treatment)
F0 Females which were not pregnant: Days 24-25 after mating
F0 Females failing to produce viable litter: Day 26 after mating
F0 Female whose litters died before Day 13: On day 2 of lactation (when the offspring missed).
F0 Females killed at termination: Day 14-16 of lactation

Method of Sacrifice:
F0 Animals: By intraperitoneal injection of sodium pentobarbital. Each animal was subsequently exsanguinated.

Gross Pathology:
Premature decdents:


Scheduled termination:
F0 Males:
Blood sampling required
Necropsy, macroscopic examinations and organs /tissues retained as follows:
- Males selected for FOB and clinical pathology: see Table B "Pathology procedures for the five males and the five lactating F0 females with a surviving litter per group at scheduled termination"
- Remaining males: See Table C "Pathology procedures for remaining F0 males and females per group"

F0 Females:
Blood sampling required
Necropsy, macroscopic examinations and organs /tissues retained as follows:
- The number of uterine implantation sites counted and recorded
- Terminal vaginal smear
- Females selected for FOB and clincial pathology: see Table B "Pathology procedures for the five males and the five lactating F0 females with a surviving litter per group at scheduled termination"
- Remaining females: See Table C "Pathology procedures for remaining F0 males and females per group"

F0 Females which were not pregnant and failied to litter:
Blood sampling
Necropsy, macroscopic examinations and organs/tissues retained as specified in Table C "Pathology procedures for remaining F0 males and females per group"
Number of implantation sites confirmed after the uterus was placed in an aqueous solution of ammonium sulfide.
Evaluation of mammary tissue and retained.
Terminal vaginal smear

F0 Females whose litter died before Day 13 lactation:
Blood sampling required
Necropsy, macroscopic examinations and organs/tissues retained as specified in Table C ""Pathology procedures for remaining F0 males and females per group"
Evaluation of mammary tissue and retained.
Terminal vaginal smear

F1 offspring on Day 4 of age:
Assessment of stomach for milk content. Offspring with no clinical observations discarded without examination.

F1 offspring on Day 13-15 of age:
Thyroid gland was retained from one male and one female in each litter which blood was collected for thyroid hormone analysis.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Abnormalities retained in appropriate fixative.

Organ Weights: Yes - Groups 1 - 4: Five F0 males and five lactating F0 femals with a surviving litter
Adult animals: See Table B and Table C
Data collection: For bilateral organs, left and right organs were weighed together.

In group 2 (100 mg/kg/day) organs were weighed in six females instead of five.

Fixation:
Fixatives:
Standard – 10% Neutral Buffered Formalin.
Testes and epididymides: modified Davidson’s fixative.
Eyes: Davidson’s fixative.

HISTOLOGY:
Processing (Table B): The F0 five males and five lactating females with a surviving litter selected in Groups 1 and 4 at scheduled termination.
Processing - Abnormalities: All adult animals
F0 Females which were not pregnant, failed to litter and whose litter died before Day 13 of lactation: Mammary tissue
Routine staining: 4-5 µm sections stained with hematoxylin and eosin, except testes which are also stained with periodic acid-Schiff

Pathology:
Light microscopy:
Groups 1 and 4: Five F0 males and five lactating F0 females with a surviving litter.
Data Recording
Ovaries: qualitative evaluation of one section from each ovary.
Testes: detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Postmortem examinations (offspring):
NECROPSY
F1 Offspring :
Selected offspring for thyroid hormone analysis – Day 4 of age
Selected offspring for thyroid hormone analysis - Day 13 of age
Remaining offspring – Day before female sacrificed

Method of sacrifice:
F1 Offspring: Selected for thyroid hormone sampling: Cardiac puncture. Death was assured with sodium pentobarbital injection.
All other offspring: injection of sodium pentobarbital.

Gross Pathology:
Premature decdents:
F1 Offspring: External macroscopic examination with an assessment of stomach for milk content, no thyroids to be retained.

Scheduled termination:
F1 offspring on Day 4 of age:
Assessment of stomach for milk content. Offspring with no clinical observations discarded without examination.

F1 offspring on Day 13-15 of age:
Thyroid gland was retained from one male and one female in each litter which blood was collected for thyroid hormone analysis.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Abnormalities retained in appropriate fixative.
Statistics:
Data types:
The following data types will be analyzed at each time point separately at the test facility, unless indicated otherwise:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods (males on pretest to be performed by PI)
Blood chemistry and hematology
Hormone analyses
Organ weights, both absolute and adjusted for terminal body weight
Grip strength and motor activity (to be performed by PI)
Estrous cycles (to be performed by PI)
Pre-coital interval (to be performed by the PI)
Mating performance and fertility (to be performed by PI)
Gestation length (to be performed by the PI)
Litter data (litter size, offspring survival, offspring body weight)
Ano-genital distance, average for each litter adjusted for litter average pup body weight (to be performed by the PI)
Number of nipples/areolae in male pups counted on post natal Day 13 (to be performed by the PI)

Methods:
The following comparisons were performed:
Group 1 vs. 2, 3 and 4
For categorical data, the proportion of animals was analyzed for each treated group (as appropriate) versus the control group.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. Treated groups were compared to control using Williams' test.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test.
For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Reproductive indices:
Pre-coital interval: Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days.

Mating performance and fertility:
Percentage mating: Number of females mating / Females_paired x 100
Conception rate: Number females achieving pregnancy / Females_mated x 100
Fertility index: Number females achieving pregnancy / Females_paired x 100

Gestation length: number of days from mating to the start of parturition (inclusive), with half a day added where parturition started overnight.
Gestation index: Number of live litters born / number pregnant x 100
Offspring viability indices:
Survival indices (%): Individual litter values calculated for:
Post-impantation survival: Total number offspring born / Total number uterine implantation sites x 100
Live birth index: Number live offspring on Day 1 after littering / Total number of offspring born x 100
Viability index: Number live offspring on Day 4 (or 6) / Number live offspring on Day 1 (or 4) after littering x 100
Lactation index: Number live offspring on Day 13 after littering / Number live offspring on Day 4 (after selection for thyroid hormone bleed) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Dark feces were recorded in males and females from day 2 of treatment to the end of study at 1000 mg/kg/day and from day 5 of treatment to the end of study at 330 mg/kg/day. These signs were considered related to the test item color.
No other signs considered related to treatment with the test item were observed during the study.
Mortality:
no mortality observed
Description (incidence):
No mortality has been recorded.
Female no. 76 at 100 mg/kg/day was sacrificed on gestation day 25 and females 81 at 100 mg/kg/day and 86 at 330 mg/kg/day on gestation day 24 as they did not give birth. Females nos. 81 and 86 were found to be not pregnant and female no. 76 had two fetuses in the uterus (failed to litter).
Female no. 105 at 0 mg/kg/day was sacrificed on day 2 of lactation after losing its litter.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Lower body-weight gain was observed in males on the following treatment periods: days 8-15 and 29-34 at 330 mg/kg/day and days 29-34 at 1000 mg/kg/day. Even though these differences are statistically significant, the percentage with respect to the control group is lower than 10% and therefore the differences are considered of no toxicological relevance.
No relevant differences were observed in females at any dose treated group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Treatment with test item resulted in no effects on food consumption.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Although no dose-effect relation was observed in hematology or coagulation, significantly lower mean values compared to Control were recorded in prothrombin time in males at 1000 mg/kg/day after 34 treatment days (21.6 sec vs 22.7 sec). This difference with respect to Control is minor and values lower than 21.6 sec were observed at 330 mg/kg/day; therefore this is attributed to the lower standard deviation recorded at 1000 mg/kg/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry after 5 weeks of treatment in males and on day 14 or 16 of lactation in females revealed lower values in alanine transferase, aspartate aminotransferase and biliary acids at all treated groups and lower creatinine kinase values at 100 and 1000 mg/kg/day.
In addition, lower creatinine levels and higher triglyceride values were recorded in males from all treated groups. The differences in glucose values recorded in females from the high dose group are attributed to the high SD observed in control animals.
There was a higher increase in sodium and chloride values in males and females at 330 and 1000 mg/kg/day with respect to control animals. Lower values in phosphor and calcium were also observed in females at 1000 mg/kg/day. However, these differences were considered not relevant, as no dose-effect relation was observed and given that these mean values were within the normal range of variability observed in animals under similar conditions.
Statistically significant differences observed in protein electrophoretogram at the test item administered groups compared to Controls were considered minimal and were therefore attributed to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity assessment conducted during week 5 in males and between days 7-8 of lactation in females revealed no relevant findings. Male no. 2 failed blink and pupillary closure reflexes of left eye.
There were no relevant effects observed in the grip strength in females. However, males treated at 1000 mg/kg/day had values higher than the control group in both limbs. The differences observed were devoid of any toxicological significance.
The motor activity assessment conducted during week 5 in males and between days 7-8 of lactation in females showed no dose-related significant differences in the test-item-administered females at any time period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All the histological findings at termination were considered to be incidental and unrelated to treatment.
There was no histological correlate for the increased adrenal gland weights in males given 1000 mg/kg compared with controls.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES:
Analyses of samples for thyroxine (T4) obtained from F0 study males and the F1 offspring on Day 13 of age (male and female pups) did not reveal any differences attributable to treatment and there was no evidence of growth effects on the offspring supporting the conclusion that there is no effect on thyroid function.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Before treatment start, all females showed regular cycles. However, one female (no. 105) at 0 mg/kg/day, one female (no. 76) at 100 mg/kg/day and one female (no. 82) at 330 mg/kg/day showed irregular cycles during treatment. Given the frequency observed, it is considered not to have any toxicological relevance.
At the end of lactation phase, all reproductive females showed diestrus with the exception of two females (nos. 63 and 68) at 0 mg/kg/day and one female at 1000 mg/kg/day (no. 92) which showed proestrus on day 14 of lactation and one female at 100 mg/kg/day (no. 73), which started cycling on day 12 of lactation.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in the examined males treated at 1000 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the pre-coital interval; all animals mated within four days of mating at the first estrus.
The duration of the gestation of females treated with the test item was similar to that of the control group. Female no.76, which did not litter, was sacrificed on day 25 of pregnancy and showed two fetuses in the uterus.
Mating performance was 100% in all test-item groups and conception and fertility were 90% at 330 and 1000 mg/kg/day and 100% at 0 and 100 mg/kg/day. This slight difference is not considered relevant as it is within the historical control data.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive/developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect of toxicological relevance on estrous cycle, pre-coital interval, mating performance, fertility or gestation length

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed among the F1 litters that could be clearly attributable to parental treatment with the test item.
Clinical signs recorded in some of the offspring were related with the lack of maternal care (such as little or no milk present in the stomach or pallor), not due to a direct test-item effect in pups.
In the control group, litter no 66 had three pups with changes on tail due to maternal biting: pup 1 with partial absence of the appendage, pup 2 with black spot at distal region and pup 9 with hind paw wound. Pup 7 from female 70 also had dark nose.
Little or no milk present in stomach was observed in pup 9 (litter 68) at the age of 4 and 5 days and in pup 1 (litter 105) on day 1. This last pup was also pale, thin and cold to the touch and finally it was devoured.
At 100 mg/kg/day, little or no milk present in stomach was observed in pup 9 (litter 73) on day 1 of age and in pup 7 (litter 72) on days 7 to 9 of age, which had been devoured.
At 330 mg/kg/day, little or no milk present in stomach was observed in pup 15 (litter 82) on day 1 of lactation and finally it was devoured.
At 1000 mg/kg/day, pup 1 (litter 96) was devoured after showing thinness on day 2 of lactation.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 Survival Indices, Litter Size and Sex Ratio :
No relevant differences were observed in post implantation losses, live birth index, and viability index on days 4, 7 and 13 of lactation. However the mean of total of pups littered (dead and alive) and the mean of live pups on days 1 and 4 at 330 and 1000 mg/kg/day were slightly lower than in the control group. The differences were only significant at 1000 mg/kg/day.
Litter no 105 from the control group was sacrificed on day 2 of lactation after losing its litter.
No test-item-related effects were observed on sex ratio.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect in the offspring body weight at 100 mg/kg/day.
There was a dose-related increase in offspring body weight of female and male pups at the doses of 330 and 1000 mg/kg/day. The differences observed are statistically significant (see differences (%) with respect the control group in the table in any other informaiton on results section).
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There was no effect in the mean ano-genital distance and weight adjusted to body weight in males. However, in female offspring, the adjusted ano-genital distance at 1000 mg/kg/day was slightly lower due to the higher body weight offspring observed.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no effects in male nipples.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings attributable to maternal treatment with test item on the offspring that died prior to the scheduled termination or among the offspring killed on days 4 or 13-15 of age.
Two pups (2M and 9F) from litter 66 (Control group) had absence of distal part of tail. This observation was previously noted as clinical sign.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect of toxicological relevance in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Offspring Body Weights:

Group

Sex

Day 1

M

 

F

Day 4

M

 

F

Day 7

M

 

F

Day 13

M

 

F

0 mg/kg/day

5.81 g

5.45 g

8.62 g

8.16 g

13.84 g

13.37 g

26.36 g

25.47 g

100 mg/kg/day

-2%

1%

-2%

0%

0%

1%

2%

3%

330 mg/kg/day

9%

11%

12%

13%

19%

18%

18%

19%

1000 mg/kg/day

9%

12%

16%

19%

25%

26%

22%

24%

Applicant's summary and conclusion

Conclusions:
The effects of oral (gavage) administration of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide to Wistar rats receiving 100, 330 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:
The No Adverse Observed Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:The No Adverse Observed Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect of toxicological relevance on estrous cycle, pre-coital interval, mating performance, fertility or gestation length, or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.
Executive summary:

Introduction:

The purpose of this study was to assess the general systemic toxic potential of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by gavage administration for at least 5-8 weeks.

Three groups of 10 male and 10 female rats receivedReaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxideat the doses of 100, 330 and 1000 mg/kg/day. Males were treated continuously for two weeks before pairing up to necropsy, after 35-36 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 13-15 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13-15 of lactation (the day before the corresponding female was killed). F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, Arachis oil.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, thyroid hormone, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight, ano-genital distance, nipple/areolae count and macropathology were also assessed for all offspring.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, thyroid hormone, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight, ano-genital distance, nipple/areolae count and macropathology were also assessed for all offspring.

Results:

The study results can be summarized as follows:

-         Preparation of all formulations was considered correct considering the acceptance ranges as formulation results demonstrated that all formulations were within the necessary acceptance range for the nominal concentration and coefficient of variation.

-         No mortality was recorded.

-         Administration of test item at 100, 330 or 1000 mg/kg/day was considered not to have caused any changes of toxicological relevance in clinical condition, body weight, food consumption, motor activity, sensory reactivity or grip strength.

-         Black feces due to the test item color were recorded during treatment in all animals treated at 330 and 1000 mg/kg/day.

-         There were no relevant effects on hematology or coagulation.

-         Although a dose-response effect was not recorded, the hepatic enzymes, CK and biliary acid values of the animals treated with the test item showed significant differences, with mean values lower than those seen in the Control group. In addition, mean sodium and chloride values higher than those observed in Control were also found in animals at 330 and 1000 mg/kg/day.

-         T4 analyses of samples in Main study males and F1 offspring on day 13 did not reveal any differences that could be attributable to treatment.

-         There were no macroscopic findings that could be considered test-item-related.

-         Adrenal and thyroid weight increase was observed in males at 330 and 1000 mg/kg/day. There was an increase in thymus and spleen weights in females from all treated groups.

-         Histopathology revealed no changes that were considered related to treatment withthe test item.

-         All females allocated to the study showed regular 4-day estrous cycles before treatment. The three females at 0, 100 and 330 mg/kg/day, which showed irregular cycles during treatment, achieved their pregnancy. At termination, 2/9 females at 0 mg/kg/day, 1/8 at 330 mg/kg/day and 1/10 at 1000 mg/kg/day had recovered estrous cycle.

-         No effect was observed in relation to the number of implantations,post implantation, survival indices,litter size or sex ratio.

-         Offspring body weight increased at 330 and 1000 mg/kg/day affecting both sexes. No differences in clinical signs, ano-genital distances, nipples count orexternal macroscopic examination at sacrificewere observed.

Conclusion:

The effects of oral (gavage) administration of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide to Wistar rats receiving 100, 330 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:

The No Adverse Observed Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:

The No Adverse Observed Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect of toxicological relevance on estrous cycle, pre-coital interval, mating performance, fertility or gestation length, or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Categories Display